Elevated T cell motility in the red pulp after PD-1 blockade at an early stage of exhaustion. (A and B) Mean track velocities (µm/min) of CD8⁺ P14 and CD4⁺ SMARTA cells were quantified in the splenic white (A) and red (B) pulp at day 7 after infection (n = 4 mice). 16 h before imaging, anti–PD-1 or isotype control antibodies (500 µg) were injected intravenously. Asterisks denote statistically significant differences (P < 0.05). Each dot represents the track of an individual T cell. Horizontal black bars denote the mean of each group. (C) Frequency distributions (5 min bins) show the average arrest duration of P14 cells in the splenic red pulp 16 h after injection of anti–PD-1 (224 cells analyzed) or isotype control antibodies (Arm, n = 219 cells analyzed; CL13, n = 261 cells analyzed; n = 4 mice). Numbers above the black arrows denote the mean of each group. Relative to the isotype control group, a statistically significant decrease (P < 0.05) in mean arrest duration was noted after injection of anti–PD-1 into CL13-infected mice. No difference was observed between Arm mice treated with isotype control antibody and CL13 mice treated with anti–PD-1. See corresponding Video 4. Data are representative of four independent experiments.